ION EXCHANGE CHROMATOGRAPHY IN FOOD ANALYSIS (Báo cáo khoa học)

HCM UNIVERSITY OF TECHNOLOGY
FACULTY OF CHEMICAL ENGINEERING
--&&&--

ESSAY TO SUBJECT OF APPLICATION OF CHROMATOGRAPHIC METHODS
IN FOOD ANALYSIS

TOPIC
ION EXCHANGE CHROMATOGRAPHY IN FOOD ANALYSIS

Supervisor:

Dr. Nguyễn Thị Lan Phi

Performer:

NGUYỄN VĂN TÚ

- 51305919

Ho Chi Minh City, 2016
TABLE OF CONTENTS


INTRODUCTION
There are a number of chromatographic methods utilized in food analysis,
including Gas Chromatography, High Performance Liquid Chromatography, Size
Exclusion

Chromatography,

etc.

Ion

Exchange

Chromatography

or

Ion

Chromatography is a technique rather commonly used in many fields in industry, and
it is of many applications in food analysis in particular.
Together with the current developments of continuous detectors, Ion
Chromatography is more and more useful in food analysis, especially corporated with
modern chromatographic techniques. Although there have been only limited
applications of Ion Chromatography in food industry, today it appears to be a hopeful
technique as a simple, unexpensive method for food industry, also for other ones.

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CONTENTS
I. ION EXCHANGE CHROMATOGRAPHY (IEC or IC)
1. Ion exchange chromatography
Ion

exchange

chromatography

is

a chromatography technique

that

separates ions and polar molecules based on their affinity to the ion exchanger.
Analytes often used in IC are large proteins, small nucleotides, and amino acids. IC is
often used in protein purification, water analysis, and quality control. The watersoluble and charged molecules such as proteins, amino acids, and peptides bind to
oppositely charged stationary phase by forming covalent bonds. The equilibrated
stationary phase consists of an ionizable functional group where the targeted
molecules of a mixture to be separated and quantified can bind while passing through
the column. [1]
The history of IC primarily began between 1935-1950 through the Manhattan
project that applications and IC were significantly extended. IC was originally
introduced by two English researchers, agricultural Sir Thompson and chemist J T
Way. It was in the fifties and sixties that theoretical models were developed for further
understanding and it was not until the seventies that continuous detectors were

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utilized, giving the way to the development from low-pressure to high-performance
chromatography. Not until 1975 was "ion chromatography" established as a name of a
technique, and was thereafter used as a name for marketing purposes.

Fig 1. Some critical timelines in the history of IC

2. Classification
Acording to the analytes, IC is often categorized into two main types, namely
Anion and Cation Chromatography. Cation exchange chromatography is used when
the desired molecules to separate are cations, and an anion exchange chromatography
is to separate anions meaning that the beads in the column contain positively charged
functional groups to attract the anions.
Similarly, the stationary phase of IC (often called IC media or IC exchanger) is
also divided into Anion and Cation Exchager. Anion Exchanger is used for Anion
Chromatography whreas Cation Chromatography uses Cation Exchanger for
separating the targeting molecules.

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Fig 2. Commonly used IC media [2]

II. PRINCIPLE OF IC
1. Net surface charge and pH
IC separates molecules on the basis of differences in their net surface charge.
Molecules vary considerably in their charge properties and will exhibit different
degrees of interaction with charged chromatography media according to differences in
their overall charge, charge density, and surface charge distribution. The charged
groups within a molecule that contribute to the net surface charge possess different
pKa values (acid ionization constant) depending on their structure and chemical
microenvironment.
Since all molecules with ionizable groups can be titrated, their net surface
charge is highly pH dependent. In the case of proteins, which are built up of many
different amino acids containing weak acidic and basic groups, net surface charge will
change gradually as the pH of the environment changes, that is, proteins are
amphoteric. Each protein has its own unique net charge versus pH relationship which

can be visualized as a titration curve. This curve reflects how the overall net charge of
the protein changes according to the surrounding pH. [2]

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Fig 3. Theoretical titration curves, showing how net surface charge varies with pH

IC takes advantage of the fact that the relationship between net surface charge
and pH is unique for a specific analyte. In an IC separation, reversible interactions
between charged molecules and oppositely charged IC media are controlled in order to
favor binding or elution of specific molecules and achieve separation. A certain
molecule that has no net charge at a pH equivalent to its isoelectric point (pI) will not
interact with a charged medium. However, at a pH above its pI, an analyte will bind to
a positively charged medium or anion exchanger and, at a pH below its pI, an analyte
will bind to a negatively charged medium or cation exchanger. In addition to the ion
exchange interaction, other types of binding can occur, but these effects are very small
and mainly due to van der Waals forces and nonpolar interactions.
2. Principle in IC separation
An IC medium comprises a matrix of spherical particles substituted with ionic
groups that are negatively or positively charged. The matrix is usually porous to give a
high internal surface area. The medium is packed into a column to form a packed bed.
The bed is then equilibrated with buffer which fills the pores of the matrix and the
space among the particles. [2]
2.1. Equilibration
The first step is the equilibration of the stationary phase to the desired start
conditions. When equilibrium is reached, all stationary phase charged groups are

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bound with exchangeable counterions, such as chloride or sodium. The pH and ionic
strength of the start buffer are selected to ensure that, when sample is loaded, analytes
bind to the medium and as many impurities as possible do not bind.
2.2. Sample application and wash
The second step is sample application and wash. The goal in this step is to bind
the target molecules and wash out all unbound one. The sample buffer should have the
same pH and ionic strength as the start buffer in order to bind all charged target
molecules. Oppositely charged items bind to ionic groups of the IC medium, becoming
concentrated on the column. Uncharged items, or those with the same charge as the
ionic group, pass through the column at the same speed as the flow of buffer, eluting
during or just after sample application, depending on the total volume of sample
loaded.
2.3. Elution
When all the sample has been loaded and the column washed with start buffer
so that all nonbinding molecules have gone out of the column, conditions are altered in
order to elute the bound analytes. Most frequently, analytes are eluted by increasing
the ionic strength (salt concentration) of the buffer or, occasionally, by changing the
pH. As ionic strength increases the salt ions (typically Na + or Cl-) compete with the
bound components for charges on the surface of the medium and one or more of the
bound species begin to elute and move down the column. The molecules with the
lowest net charge at the selected pH will be the first ones eluted from the column as
ionic strength increases. Similarly, the components with the highest charge at a certain
pH will be most strongly retained and will be eluted thereafter. The higher the net
charge of the target molecules, the higher the ionic strength that is needed for elution.
By controlling changes in ionic strength using different forms of gradient, components
are eluted differently in a purified, concentrated form.

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Fig. 4. Description of steps in IC separation

2.4. Regeneration
A final wash with high ionic strength buffer regenerates the column and
removes any molecules still bound. This ensures that the full capacity of the stationary

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phase is available for the next run. The column is then re-equilibrated in start buffer
before starting the next run.
Alternatively, conditions can be chosen to maximize the binding of
contaminants to allow the target analytes to first pass through the column to be
collected.
3. Resolution
The resolution of an IC separation is a representation of the degree of
separation between the peaks eluted from the column (the selectivity of the medium),
the ability of the column to produce narrow, symmetrical peaks (efficiency) and, of
course, the amount (mass) of sample applied. These factors depend upon practical
issues such as matrix properties, binding and elution conditions, column packing, and
flow rates.
Resolution (Rs) is defined as the distance between peak maxima compared with
the average base width of the two peaks. Rs can be determined from a chromatogram,
as shown in Figure 5. [2]

Fig. 5. Theoretical determination of Rs in IC separation [2]

Rs gives a measure of the relative separation between two peaks and can be
used to determine if further optimization of the chromatographic procedure is

necessary.

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If Rs = 1.0 then 98% purity has been achieved at 98% of peak recovery,
provided the peaks are symmetrical and approximately equal in size. Baseline
resolution requires that Rs ≥1.5. At this value, peak purity is 100% (Fig. 6).

Fig. 6. Separation result with different Rs, showing if further optimization is needed

4. IC system
Similar to some other chromatography systems, IC has some fundamental
components as the following figure:

Fig. 7. A typical IC system

The eluent generator is used to generate the proper buffer for analysis, follwed
by the separation collumn with the charged stationary phase. The analysis data is
obtained by combination with the detector connected to a screen.
III. PROS AND CONS OF IC

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IC is a very powerful separation technique that is used not only for preparative
chromatography but also for analytical chromatography. However, like all other
chromatography modes, IC does have some limitations.
One of the main disadvantages of IC is its buffer requirement: because binding
to IC media is dependent on electrostatic interactions between analytes of interest and

the stationary phase, IC columns must be loaded in lowsalt buffers. For some
applications, this restriction may require a buffer exchange step prior to IC analysis.
IC, unlike some other chromatography methods, also permits high flow rates,
which in some cases can be crucial to the recovery of active protein. Finally, a
limitation of weak ion exchangers is their pH dependence. When working outside of
their optimal pH range, these resins rapidly lose capacity, and more importantly,
resolution as table below. [3]
PROS

CONS

- Permits high flow rate

- Sample must be loaded at low ionic strength

- Concentrates samples

- Clusters of positively charged residues can
cause a net negatively charged protein to bind a
cation exchanger, and vice versa

- High yield

- Small changes in pH can greatly alter binding
profile of IC resin

-Buffers are non-denaturing

- Particle size greatly influences resolution

IV. APPLICATIONS
IC is a powerful technique in a number of field, such as environment analysis,
water treatment, pharmaceutical and drugs analysis and food analysis. IC has been
utilized in environment and water treatment which the charged resins was used to
separate metallic ion from the natural resources. Though applications in
pharmaceutical, drugs and food analysis are less than two fields above, IC gradually
becomes hopeful technique corporated to other chromatography methods. [4-6]
To illustrate, a remarkable investigation conducted in 2005 by C. Gu´erinDubiard et al. separated useful proteins in hen egg white into separate fractions. Due to
the dependence of IC upon pH, researchers changed the pH to get individual

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components that have the different pI value. This separation procedure was depicted as
following figure:

Fig. 8. Procedure of hen egg white separation [7]

The analytical results and the confirmation of analytes was determined by
HPLC to delight the separation efficiency and the accuracy of this method (Fig. 9).

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Fig. 9. HPLC analysis of hen egg white separation, showing the method efficiency

Based upon the HPLC analysis, proteins in hen egg white was separated
successfully with the high efficiency. This paper also showed the importance of

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changing pH in IC in which flexibly changing pH of buffer can completely purify the
analytes of interest in a mixture.

CONCLUSION

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Ion exchange chromatography is a technique that separates charged molecules
from the mixture basing upon their net surface charge at specific pH of the
surroundings. Anion exchange chromatography uses the anion exchagers formed by
functionalized the stationary phase with cationic groups for separation of anion
analytes. By contrast, cation exchage chromatography separates the cationic molecules
from the mixture while they pass through the collumn filled by negatively charged
matrices.
IC is a useful technique used in a wide number of fields, especially in
environment and water treatment. In food industry, together with the developments of
continuous detectors currently, IC has been utilized with other chromatographic
methods for separation of charged molecules such as proteins, amino acids and
peptides. Like other modes of chromatography, IC has advantages and disavantages
which can be noted to enhance the efficiency and resolution of this method.

REFERENCES

1.

Wikipedia. Ion Chromatography. Available from:
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2.
3.
4.
5.
6.
7.

Healthcare, G., Ion Exchange Chromatography: Principles and Methods,
GE Healthcare: GE Healthcare.
BioRad, Ion Exchange Chromatography: Applications & Technologies
BioRad: BioRad.
Jackson, P.E., Ion Chromatography in Environmental Analysis.
Encyclopedia of Analytical Chemistry, 2000: p. 2779–2801.
Rohrer, L.B.a.J.S., Application of IC for pharma and biological products.
A JOHN WILEY & SONS, INC., PUBLICATION, 2012.
MICHALSKI, R., Industrial applications of ion chromatography.
CHEMIK, 2014. 68: p. 478-485.
al, C.G.e.-D.e., Hen egg white fractionation by ion-exchange
chromatography. Journal of Chromatography A, 2005. 1090: p. 58-67.

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